Finally, comparisons were made of pHPP.1 content of several urines run by this method and the one presently described. Table I11 shows the effect of Lloyd’s reagent on pure standards of p H P P h . Table IV compares the procedure outlined above for pHPPA (Method A) with one utilizing Lloyd’s reagent folloiied by Brigg’s reaction (Method B). It is apparent that Method B suffers inherently from two compensating errors. Lloyd’s reagent adsorbs pHPP.1. In most instances a reducing hubstance other than pHPPA remains in the urine even after treatment with the reagent and contributes to the final color. The constitution of any particular urine \Till determine to IThat extent the two errors compensate and a correct answer is largely fortuitous. ACKNOWLEDGMENT
The authors express their appreciation for the technical assibtance rendered by Julia Nitchell. Thanks are due also t o Gloria Senienuk who contributed the paper Chromatographic work.
Table IV. pHPPA in Urine as Determined with Brigg’s Reaction (8) Compared to That Obtained through the Procedure Described in This Paper (A)
Sample DJI HK LL DH HA1
RT’
Millierams DHPPA excYetedli4 hours Method A Method B 10 4 6 0 9 8 15.0 21.0 14.5
41 77 87 72 131 132
LITERATURE CITED
(1) Billek, G Monatsch. 92, 335 (1961). (2) Briggs, 2. H., Harvey, N., Life Sciences 1, 61 (1962). ( 3 ) Bucher. T.. Kirbereer. E.. Biochim. Bzophys. Acta 8 , 401 fi952). ’ (4) Doy, C., Nature 186, 529 (1960). (5) Ewald, W.,Hubener, H., 11~uturu;iss. 48, 720 (1961). (6) Gros, H., Kirberger, E. J., Klin Wochschr. 32, 115 (1954). (7) Henning, T’., Amnon, R., 2. Physiol. Chem. 306, 221 (1957). ( 8 ) Humbel, R., M e d . Lab. 17, 68 (1964).
(9) Knox, W. E., Goswami, hl., J . Biol. Chem. 235, 2662 (1960). (10) Knox, W. E., Pitt, B., Ibid., 225, 675 (1957). (11) Levine, S., hlarples, E., Gordon, H., J . Clin. Invest. 20, 199 (1941). (12) Lin, E., Pitt, B., Civen, &I., Knox, W. E.. J . Biol. Chem. 233. 668 (1958). (13) Nedes, G., Biochem.’ J . 26, 917 (1932). (14) Nishimura, N., Maeda, K., Yasui, S . , Okamoto, H., Natsunaka, hl., Teshina, H., Arch. Derm. 83, 644 (1961). (15) Yonouchi, Y., il’aika Hoken 7, 610 (1960); C. A . 5 5 , 11615f (1961). (16) Painter, H., Zilva, S., Biochem. J. 41, 520 (1947). (17) Pitt, B., Nature 196, 272 (1962). (18) Wieland, H., Ann. Chem. 436, 229 (1924). (19) Williams, C., Anal. Biochem. 4, 423 (1962). (20) Woolf, L. I., “Advances in Clinical Chemistry,” 1-01. 6, p. 187, H.Sobotka
and C. Steward, eds., Academic Press,
New York, 1963. (21) Woolf, L. I., Edmunds, N. E., Biochem. J . 47, 630 (1950). (22) Schwartz, K., Arch. Biochem. Biophys. 92, 168 (1961).
RECEIVED for review December 22, 1964. Resubmitted August 30, 1965. Accepted September 29, 1965. Work supported by C. S. Public Health grant AM-05776.
Direct Radiochemical Determination of Lead-210 in Bone HENRY G. PETROW and ARTHUR COVER lnsfitute o f Environmenfal Medicine, New York University Medical Center, New York,
b A procedure for the radiochemical determination of lead-210 in bone ash is based on the solvent extraction of a lead bromide complex into a quaternary amine. Lead-210 is detected, after a suitable ingrowth period, b y beta-counting its bismuth2 10 daughter. Ash samples up to 30 grams can b e routinely analyzed, and as little as 2 X 1 O - I 2 curie of lead-2 10 can be determined. Interference from other elements, both stable and radioactive, is slight. The procedure is applicable to materials other than bone ash.
B
of the extremely low energy of its beta emission, low concentrations of lead-210 in environmental samples are frequently determined by measurement of polonium-210 ( 1 ) . If this technique is to be reliable, the sample niust first be freed of any polonium-210 initially present. S o lead loss can be tolerated during this separation. After the initial polonium separation, the lead-bearing sample must be set aside for months to allow fresh ingronth of polonium-210 from lead-210 present in ECAUSE
the sample; then a second polonium separation must be performed. Obviously, this is a long, time-consuming process, and is subject to error if the polonium-recovery process is not precise for samples of varying composition. As an alternative to this procedure, lead-210 can be separated and allowed to age for several days, and the 5.0-day bismuth-210 daughter beta-counted. A lead-210 assay can be completed in a few days. Lead recovery can be accurately determined through use of known amounts of carrier lead. If the lead-210bismuth-210 level is too low to permit accurate beta counting, the separated sample can be conveniently stored for polonium-210 ingrowth, since the lead sample, as initially separated, is completely free from polonium present in the original sample. Furthermore, variations in polonium-210 recovery can be minimized, since polonium will always be deposited from an identical matrix, after the lead sample has been dissolved in hydrochloric acid. I n this way, advantage can be taken of the rapidity of the lead-210-bismuth-210 technique and, if necessary, of the ex-
N. Y. treme sensitivity of the polonium method. The procedure is based upon the extraction of a lead bromide complex ion into a quaternary ammonium bromide (.iliquat 336). As an indication of the effectiveness of lead bromide extraction from phosphate solution, 1 gram of lead, as the nitrate, dissolved in 50 ml. of 85%;’,phosphoric acid, is completely extracted into 50 ml. of 30% Aliquat 336 bromide in toluene. EXPERIMENTAL
Apparatus. Omniguard low-background beta counter, Tracerlab, Inc. Aliquat 336 (General Mills Chemical Co.) , RIethyltricaprylammonium chloride, 30 volume % in toluene, washed twice with a n equal volume of 1.53i‘ hydrobromic acid. Procedure. Place up t o 100 grams of bone in a muffle furnace a t 580” t o 600” C. Maximum temperature should not exceed 600”, since higher temperatures result in the loss of lead. Heat for 12 t o 16 hours t o yield a white ash. Keigh up to 30 grams of ash, add 7 ml. of 3 X hydrobromic acid per gram of ash and, 1 ml. of VOL. 37, NO. 13, DECEMBER 1965
1659
Table 1.
PbZ1O added,
d.p.m.
Analysis of Spiked Bone
Lead PbZ1O Ash n-eight, recovery, found % d.p.m. grams 15 16.9 29 30 15 15
1450
98.1 93.1 86.7 97.9 99.8 101
1420 1470 1490 1450 1465 1470
If the beta count rate is too low to allow an accurate determination, age the sample for a known period of time, usually 3 to 6 months, and dissolve the lead sulfate in hydrochloric acid. Deposit the polonium onto a silver disk as described by Holtzman ( 1 ) and from the observed alpha count, the known lead recovery, the polonium ingrowth factor, and the polonium recovery factor, calculate the lead-210 concentration. RESULTS
0.10 -11lead nitrate solution, and heat until the ash dissolves. Transfer the qolution to a separatory funnel containing 50 ml. of Aliquat 336 and extract for 30 seconds. Discard the aqueous phase. K a s h the extract three times for 30 seconds Kith 25-ml. portions of 0 . 1 X hydrobromic acid. Discard all three wash solutions. Strip the lead from the solvent by contacting twice, for 30 seconds, with 25-m1. portions of concentrated hydrochloric acid. Combine the strip solutions in a beaker containing 1 ml. of 0.025X bismuth nitrate solution and 4 ml. of 9 J I sulfuric acid. Di>card the solvent. Evaporate the solution t o dense fumes of sulfuric acid and continue heating until the solution turns jet black from the decomposed solvent. Cool the solution, add 5 ml. of nitric acid, and heat to fumes of sulfuric acid. Cool the solution, add 25 ml. of Fater, and filter the lead sulfate precipitate onto a weighed Whatman Xo. 42 filter disk. S o t e the time of filtration. Wash the precipitate with 0.01V sulfuric acid, followed by acetone. Dry the sample to conqtant lveight at 110' C., to determine the lead sulfate recovery. Mount the filter disk on a ring mount of witable size, covering the sample n-ith a thin Mylar film. After 3 to 20 days, beta-count the sample, noting the time of counting. To correct count rate t o disintegration rate, prepare a self-absorption curve for lead sulfate, using standard lead nitiate solutions and a purified standard solution of lead-210 free of polonium-210. TO calculate the lead-210 concentration, use the expression: Lead-210, d.p.m./gram ash = c.u.m. - B c.p.m.
I' where c.p.m.
d.p.m.x
B
= =
Y
=
-__ c.p.m. =
d.p.m. TT'
t
1660
= =
W
x
(1 -
e-0.1399
sample count rate instrument background, c.p.m. fractional lead recovery, usually 0.95 to 1.0 counts per disintegration a t sample thickness rveight of sample, grams days elapsed between filtration of lead sulfate and time of counting
ANALYTICAL CHEMISTRY
The procedure was evaluated by adding a known amount of standard lead-210 solution to bone. (Table I), Next, replicate analyses of ashed steer bone were made. A total of eight 15gram samples were analyzed with a resulting average of 2.46 i 0.13 d.p.m. of lead-210 per gram of ash. Replicate analyses of a composite sample of some 60 human teeth yielded 0.61 i 0.08 d.p.m. of lead-210 per gram of ash. Each result represents the analysis of one-fourth of the dissolved sample. Finally, a sample of reindeer bone received from R . B. Holtzman was analyzed. Holtzman, using the polonium technique, determined a lead210 content of 6.4 d.p.m. per gram of bone. The authors, using the procedure described above, determined a lead210 concentration of 6.0 d.p.m. per gram of bone. DISCUSSION
In this method the diminished sensitivity of beta counting is partially overcome because of the ease with which very large samples of bone ash can be analyzed. Neither calcium nor phosphate interferes with the recovery of lead, and lead can be easily and quantitatively separated from as much as 30 grams of bone ash in a single extraction. Holtzman reports an average lead-210 content in human curie per gram bone of 0.146 X of ash ( 1 ) . At this concentration, the amount of lead-210 present is considerably above the sensitivity of the procedure, 2 x curie, if a 30-gram sample is analyzed. Interference from both stable and radioactive substances was considered. There is no interference from radium, uranium, thorium, actinium, strontium, barium, the rare earths, calcium, yttrium, polonium, iron, nickel, cobalt, aluminum, magnesium, phosphate, or sulfate. A small amount of any radiobismuth present accompanies the lead, but is effectively eliminated through use of a bismuth holdback carrier.
Mercury accompanies the lead through the extraction procedures, but is eliminated by the lead sulfate precipitation. High concentrations of perchlorate and to a lesser extent nitrate ion inhibit lead extraction. Another investigator, using the above procedure for the determination of lead-210 in rain water, found some interference from fission product antimony-125 (3). Antimony was completely co-extracted with lead, and about 1% of the antimony was stripped from the Aliquat 336. Since lead is essentially nonextractable from the strip solution, the interference from antimony should be virtually eliminated by washing the strip solution with an equal volume of 30% Aliquat 336, and antimony interference reduced by a factor of 100. This procedure is a simple and rapid method of determining lead-210 in uranium mill effluent solutions. I n this case, lead carrier is added to the effluent solution, of a maximum volume 250 ml., and the solution is made 0.5M in hydrobromic acid, which dissolves any lead sulfate precipitated, and treated in the same manner as dissolved bone. The procedure has also been applied with succeSs to food ash dissolved in hydrobromic acid. One last consideration is the stable lead content of bone. Survey of many discussions of the lead content of human bone indicates a range of 10 to 40 p.p.m. per gram of ash. At the higher level, when 30 grams of ash were used, about a 6% error would be introduced due to incorrect yield calculation. Therefore, it may be desirable t o determine lead in a portion of the disbolved bone, prior to addition of lead carrier, but in most instances, this should not be necessary. The procedure of Ilcetvicz, Holtzman, and Lucas is convenient for this purpose ( 2 ) . Steer bone contains less stable lead than human bone. The steer bone analyzed in the work reported contained less than 5 p.p.m. of lead in the ash. ACKNOWLEDGMENT
The authors thank R. B. Holtzman for much valuable information and the analyzed sample of reindeer bone, and Walter J. Schiessle for his assistance, LITERATURE CITED
(1) Holtzman, R. B,, Health Phys. 9, 385 (1963). (2) Ilcewicz, F. H., Holtzman, R. B., Lucas, H. F., Jr., ANAL. CHEM. 36, 1132 (1964). (3) Magno, P. J., Northeastern Radiolog-
ical Health Laboratory, R7inchester,
Ifass., private communication. RECEIVEDfor review June 11, 1965. Accepted September 7 , 1965.
